1. Field of the Invention
This invention pertains generally to chemical analytical and immunological testing, and particularly to processes wherein samples are analyzed by using self-operated mechanisms or devices, and more particularly to processes wherein a continuously flowing stream of a sample or carrier fluid is formed and flows into and through analysis wherein the continuously flowing stream is segmented by alternately injecting a sample, reagent or any number of fluids into a common flow path.
2. Description of the Related Art
Recent events have highlighted the need for devices that can quickly ascertain and identify the presence of harmful materials in airborne particles. There is also a more general need, beyond the biological-warfare (BW) agent detection problem, for improved methods for measuring analytes in airborne particles. For example, airborne infectious agents (bacteria, viruses) transmit many diseases of humans, other animals, or plants. Some airborne proteins and pollens cause allergies. Improved methods for characterizing aerosols would be useful for understanding atmospheric chemistry, including the sources, chemical reactions, and fates of atmospheric particles.
Here, “airborne particle” refers to both the solid particles and liquid droplets in an air sample. The analyte is the specific molecule, microorganism, or virus to be identified. For example, for biological warfare agents that are protein toxins, e.g., ricin, the toxin itself is the analyte. For BW agents that are bacteria or viruses, the analyte can be a molecule that is specific to the bacteria or virus to be detected, e.g., a protein or a DNA or RNA sequence. In this case the amount of the analyte is measured; if this amount is significantly above a noise threshold, the presence of the BW agent is inferred. For BW agents that are bacteria or viruses, the analyte can be the bacteria or virus itself.
Key objectives for some types of instruments needed for detecting BW-agents or other analytes in airborne particles are:
(a) Sensitivity. An instrument should be able to measure and identify small amounts of a BW-analyte in the particles in an air sample, because small amounts of BW agents may be lethal.
(b) Specificity. An instrument should have a very low rate of false positives, i.e., reporting a BW-analyte when it is not in the air sample.
(c) Rapid response. An instrument should have no more than a short delay between the time a BW aerosol enters the instrument and the time the instrument indicates that a BW-analyte has been identified. The sooner people know they are under attack, the sooner they can take protective measures if available, and/or try to leave the region of exposure, and/or seek medical treatment. Also, with a sufficiently rapid alert some people can avoid exposure altogether.
(d) Continuous operation. An instrument should be able to run essentially continuously for days or weeks at a time. It should run continuously because BW aerosols could appear at any time. Presently, “trigger” instruments, which run continuously but cannot identify BW-agents, are used to tell when to turn on instruments that can identify agents. If there were some “trigger” instrument that was adequate for telling when to turn on an identifier, there would be no need for an identifier. But it is difficult to imagine that any of the reagentless techniques being investigated or suggested for trigger instruments would be able to identify specific BW agents in cases where these BW agents comprise a small fraction of the total particles in a complex mixture of airborne particles.
(e) Little need for consumables. An instrument should not require large amounts of consumables (e.g., liquids, antibodies, microscope slides, filters). The more consumables required, the fewer BW-aerosol-detection instruments that can be maintained in continuous operation.
(f) Little need for operator time. If more operator time is required, fewer BW-aerosol-detection instruments can be maintained in continuous operation.
(g) Be able to separate and store particles for further analysis. It is desirable to confirm the detection of analyte using complementary techniques which may be much less rapid.
Investigators have worked for years to develop instruments and methods that are useful for detecting airborne BW agents. Samples can be collected from air using a variety of different collectors, and the collected samples can be subjected to many different types of microbiological and biochemical analyses. Therefore, the number of possible approaches is very large. Because of the importance of the problem, progress is being made, e.g., improved recognition molecules such as antibodies and aptamers for BW agents are being developed; more rapid methods of extracting DNA and RNA from spores are being explored; methods for detecting very small amounts of analytes or very small amounts of antigen-antibody reactions are being improved and new methods are being developed; improved methods of concentrating airborne particles, and collecting them from air are being developed; and instrumentation is being developed to perform the analysis in an automated fashion, for example, an automated flow cytometer has been developed for BW-agent detection.
None of these methods adequately satisfy the objectives stated above simultaneously. Some reasons for these objectives not being met simultaneously are as follows. Objectives (a) and (b) require sensitivity and specificity. To measure the amount of an analyte that is a BW agent or is indicative of a BW agent in a complex sample (collected from air or otherwise), requires the sample to be mixed with one or more liquids, termed here, “analysis liquids.” At least one of these liquids contains sensor molecules, also termed recognition molecules, that selectively bind to or interacts with the analyte. Example recognition molecules are antibodies and aptamers. Aptamers are DNA or RNA molecules that are selected for their ability to bind to the analyte. As a result of this binding of the recognition molecule to the analyte, some measurable property, e.g., fluorescence, must change according to the amount of analyte in the sample. That property is measured and the amount of analyte is inferred. Objectives (c) and (d) require continuous operation for days or weeks, and therefore continuous expenditure of consumables. Therefore, because of objective (e) limiting consumables, each measurement must require only a very small amount of consumables. In addition to the consumables used in analyzing the sample, consumables are typically expended in collecting particles from the air to be analyzed. If the particles are collected on filters or impacted on a surface, the filter or surface is a consumable unless it is cleaned, in which case whatever is used to clean it may be consumed. In typical analysis procedures for biochemical analytes in airborne particles, the airborne particles are collected into a liquid, which tends to evaporate as the sample is collected, especially if the air sample is warm and dry.
The objectives of sensitivity and specificity, suggest choosing as analytes specific DNA or RNA sequences, and this approach may be applicable for some analytes. However, objective (c) for a rapid response makes this approach not feasible for spores because 10's of minutes are required for the DNA from a spore to be extracted, amplified and detected. Also, this approach is not applicable to BW agents that do not contain DNA or RNA, such as protein toxins.
Arnold, Hendrie and Bronk (U.S. Pat. No. 5,532,140, Method and Apparatus for Suspending Microparticles, herein incorporated by reference) described a linear quadrupole (LC) with rings to control particle motion. They describe how the positions of the charged particles can be controlled by moving storage rings that encircle the LQ, and they describe how oppositely charged particles can be combined by moving them toward each other by moving the storage rings. Although the Background and the Summary of the Invention mention the problem of characterizing atmospheric, and biological warfare agent aerosols, there is nothing in the detailed description of the invention that suggests colliding an atmospheric aerosol particle with a droplet. The two droplets that collide are each generated with a piezoelectric droplet generator and a charging plate, which combination I term a charged-droplet generator (CDG). Because the two charged droplets are each generated with a CDG, one has the impression that the atmospheric particles would first be collected into a liquid, and the droplets would be generated from this liquid. That approach is valid, but would require more liquid for each particle than if each atmospheric particle of interest is collided with a single droplet, and it is susceptible to particles sticking to surfaces, etc. Arnold and coworkers (A. F. Izmailov, A. S. Myerson, S. Arnold, “A statistical understanding of nucleation,” J. Crystal Growth, 196, 234–242 (1999), especially FIG. 1 and pages 238 and 240, both herein incorporated by reference) further stated that their experiments show they “can simultaneously levitate in excess of 100 identical microdroplet particles within the same LQELT. These particles produce a periodic one-dimensional lattice.” M. D. Barnes, N. Lermer, C.-Y. Kung, W. B. Whitten, J. M. Ramsey, S. C. Hill, “Real-time observation of single-molecule fluorescence in microdroplet streams,” Optics Letters, 22, 1265–1267 (1997), incorporated herein by reference, showed that single fluorescence molecules in droplets can be detected. The droplets are generated with a droplet generator and a charging ring, a combination that comprises a charged droplet generator (CDG), and are then confined by a LQ to the axis of the LQ. Laser induced fluorescence from the single molecule, is detected as the droplet flows through a laser beam that is perpendicular to and passes through the LQ axis. In other experiments, particles as small as 1 micrometer have been shown to have trajectories that remain very near the LQ axis.
Individual droplets can be levitated and their reactions with gases or particles can be monitored. See E. James Davis and Gustav Schwieger, The Airborne Microparticle, Its Physics, Chemistry and Transport Phenomena (Springer-Verlag, Berlin, 2002), especially pp. 69–116 (with references) for electrodynamic levitators, and pp. 682–714 (with references) for measurements of chemical reactions in falling or levitated droplets. A commonly used electrodynamic levitation apparatus is termed the electrodynamic balance (EDB). It confines particles in three dimensions. C. L. Aardahl, J. F. Widmann, and E. J. Davis, in “Raman Analysis of Chemical Reactions Resulting from the Collision of Micrometer-Sized Particles,” Applied Spectroscopy, 52, 47–53 showed that two particles levitated in an EDB could be combined and the reaction between them monitored using Raman scattering.